Vascular disorders, such as acute myocardial infarction, stroke, deep vein thrombosis, pulmonary embolism, peripheral arterial thrombosis or other thrombotic diseases are caused by partial or total occlusion of the blood vessel by a blood clot. In order to restore the blood flow the clot has to removed or degraded. The degradation of blood clots can be achieved by the use of plasminogen activators. Such activators are able to convert the plasma proenzyme plasminogen into its active form, plasmin. Plasmin efficiently degrades fibrin which is the main component of the clot.
The compounds which are presently available for thrombolytic treatment may be classified into three groups. Tissue-type plasminogen activators (t-PA), urokinase-type plasminogen activators (u-PA) and streptokinase. For the treatment of thrombosis they all have certain limitations. Streptokinase is a bacterial protein and induces an immunologic response which can cause clinical problems. Both u-PA and streptokinase lack fibrin selectivity and fibrin affinity. Intravenous administration of these activators generate plasmin systemically which may cause a haemorrhagic potential and bleeding complications. Tissue plasminogen activator has a high affinity for fibrin and is only efficiently activating plasminogen when it is bound to fibrin. Thus, t-PA can efficiently degrade the fibrin clot without causing systemic plasmin effects.
Recombinant DNA techniques as well as conventional biotechnical methods have been employed in order to produce t-PA for thrombolytic therapy [European Patent application number 93619, European Patent application number 41766 and European Patent Application number 178105]. Clinical studies with recombinant t-PA have shown that efficient thrombolysis requires doses of 80-100 mg. The high doses required may be related to the fact that t-PA is rapidly cleared from the circulation by the liver and the half-life of native t-PA in man is only a few minutes [Garabedian et al. 1986, Am. J. Cardiol. 58, pp 673-679]. The short half-life also makes it necessary to administer the activator as a continuous infusion over several hours instead as a more convenient and faster bolus injection.
Therefore, attempts have been made to generate plasminogen activators with high fibrin affinity and fibrin selectivity having also an extended half-life in the blood. It is anticipated that such plasminogen activators will be effective at lower doses than what is presently used for t-PA and that bolus injection could be used instead if infusion.
Genetically modified variants of t-PA having an extended in vivo half-life have been produced by recombinant DNA techniques [European Patent Applications 88850207.7 and 242836 and International Patent Application PCT number WO 87/04722]. Animal tests with such modified t-PA molecules have shown that slower clearance is associated with a more efficient thrombolysis [Collen et al. 1988, Blood 71, 216-219;].
However, when the variants are tested in vitro for their ability to lyse human blood clots the activity is associated with a marked lag-phase [Kalyan et al. 1988, J. Biol. Chem. 263, 3971-3978]. This lag-phase reduces the activity of the variant and increases the lysis time, thus counteracting the benefits of a longer half-life in vivo.